1. Field of the Invention
This invention relates to a method for assay of glycosylated hemoglobin, and more particularly, to a homogeneous method and materials for determination of the percentage of glycosylated hemoglobin in the total hemoglobin of a blood sample.
2. Background of the Invention
Hemoglobin (Hb) is a mixture of proteins consisting of a prosthetic heme group attached to two pairs of unlike polypeptide chains, and in this disclosure, Hb refers in general to this mixture. In the major component of the mixture, conventionally terme HbA, the chains are designated .alpha. and .beta.. Total Hb, hereinafter termed Hbt, includes the major .alpha..sub.2, .beta..sub.2 fraction and several minor components having different chains, and various hemoglobin fractions formed by nonenzymatic glycosylation reactions of intact Hb molecules. As used in this disclosure, Hb and Hbt actually designate the same material. For clarity, Hbt is used to emphasize total hemoglobin, and Hb is used for general discussion which can apply to any hemoglobin fraction or combination of fractions.
The fractions of Hb are conventionally separated by chromatography. The main chromatographic fraction, making up about 90% of Hbt, is nonglycosylated and is generally referred to as HbA, and, in this disclosure, the term HbA is used to designate total nonglycosylated Hb. The major fraction of glycosylated Hb, conventionally and in this disclosure, is termed HbAlc.
HbAlc arises by reaction of a terminal valine amino group in the .beta. chain with the aldehyde group of glucose to give an unstable aldimine. Amadori rearrangement of the aldimine gives HbAlc, which is characterized by a .beta.-ketoglycoside linked to the valine amine group.
The determination of HbAlc and the percentage thereof in Hbt is important in the diagnosis of diabetes mellitus and in monitoring the treatment of diabetic patients. In nondiabetic people, the HbAlc level is generally between 4-8% of Hbt. In diabetics, the HbAlc level is 2-3 fold higher and may range up to 20% of Hbt.
A variety of methods has been proposed for assessment of HbAlc and Hbt levels in a sample of a patient's blood. Early macrochromatographic separation methods have been supplanted by microchromatography methods using ion-exchange resins as column packing. U.S. Pat. Nos. 4,270,921 to Graas, 4,389,491 to Hanamoto, 4,436,820 to Reiter and 4,407,961 to Sanders are exemplary of HbAlc determinations using ion-exchange techniques.
Other disclosures achieve separation without ion-exchange. Electrophoretic separation is described in U.S. Pat. No. 4,351,711 to Ambler. Electrochromotographic separation is disclosed in U.S. Pat. No. 4,222,836 to Kerr. U.S. Pat. No. 4,269,605 to Dean discloses complexation of HbAlc with a dihydroxyboryl reagent. In this method, separation of the complex from other Hb fractions is carried out by physical means, such as by bonding the boryl reagent to a solid phase, so that HbA can be washed away.
All existing methods for determination of HbAlc involve separation of the HbAlc from Hbt prior to measurement. These separation steps entail repeated accurate pipetting steps and are costly, time-consuming, labor intensive procedures requiring skilled technicians. There is a definite need for a simple, rapid, accurate assay which can be performed by unskilled technicians. It is toward the fulfillment of this need that this invention is directed.